Diagnosis and treatment of friedreich&#39;s ataxia

ABSTRACT

The present invention is directed to oligonucleotides based on peptide nucleic acid oligonucleotide or an equivalent oligonucleotide analogue, such as morpholino or a locked nucleic acid sequences and the use of such oligonucleotides for the dissociation of higher order structures, including triplex-helix DNA structures, in repeated sequences of DNA in Friedreich&#39;s ataxia. The dissociation of such structures may be used in the diagnosis and/or treatment of Friedreich&#39;s ataxia. Consequently, the present invention is also directed to a method for diagnosing Friedreich&#39;s ataxia and the use of peptide nucleic acid oligonucleotide or an equivalent oligonucleotide analogue, such as morpholino or a locked nucleic acid sequences in the treatment of Friedreich&#39;s ataxia. Preferably, the oligonucleotides comprise a sequence selected from the group consisting of (GAA) n , (CTT) n , (JTT) n  or a mixed (JTT/CTT) n  sequence.

TECHNICAL FIELD

The present invention is in the field of diagnosis and/or treatmentand/or prevention of Friedreich's ataxia of afflicted subjects orsubjects at risk of being afflicted. More particularly, the inventionconcerns the dissociation of higher order structures of DNA, includingtriplex structures, by oligonucleotide repeats based on peptide nucleicacids, locked nucleic acids or equivalent oligonucleotide analogues,said oligonucleotide repeats being complementary to the sequence of theexpanded sequence repeat involved in Friedreich's ataxia.

BACKGROUND ART

Friedreich's ataxia (FA), a neurodegenerative disease, is the mostcommon inherited autosomal recessive ataxia, and is caused in 98% of allcases by expansion of GAA repeats in the first intron of the frataxingene (FXN) (1). In healthy individuals the alleles may contain up to 40GAA repeats, whereas expanded alleles in FA patients can consist of 90to 1700 repeats (2). The GAA repeat expansion leads to major reductionin frataxin mRNA and low levels of the protein in mitochondria (2). Alsocarriers (heterozygous for the expanded allele) show ˜50% reduction ofmRNA and protein levels compared to normal expression, although they donot show any symptoms (1). Frataxin deficiency causes excessive freeradical production, dysfunction of Fe—S center containing enzymes, andprogressive iron accumulation in mitochondria (3).

Friedreich's ataxia is a deadly disease and affects people at an earlyage. Today, there is not any way to cure or prevent the disease andcurrent therapy can only treat the symptoms. The number of expanded GAArepeats in FA is directly correlated to the age-of-onset and severity ofthe disease.

Expanded GAA repeats form an intramolecular triple-helix (triplex),so-called H-DNA, (FIG. 1) in supercoiled plasmids isolated from E. coli(4). Several models representing the triplex structures formed atexpanded GAA repeats are proposed, and direct evidence for a pyrimidinemotif H-DNA structure at pathological GAA expansions in vitro hasrecently been provided (5). Also, formation of a higher order structurenamed “sticky DNA” has been observed in frataxin GAA repeats-containingplasmids using gel electrophoresis and atomic force microscopy (4). Themolecular structure of sticky DNA is not resolved; however, currentevidence demonstrates that sticky DNA forms as one long intramoleculartriplex structure or by the association of two triplexes.

Structural properties of GAA repeats may affect the stability of therepeat length as well as expression of frataxin (6). Long GAA repeatswere shown to stall replication in vivo in Saccharymyces cerevisiae (7)and inhibit transcription in vitro and in transfected cells (8).

The observed effects on DNA replication and transcription are dependenton the length and orientation of the GAA repeats in plasmids, whichcorrelate with formation of the specific DNA structure (H-DNA). Finally,the GAA repeats are associated with a pattern of DNA methylation andhistone acetylation in the adjacent regions and the formation ofsilenced chromatin. The presence of H-DNA and higher order structureswithin the GAA repeats is believed to recruit chromatin-remodelingprotein complexes that maintain a close chromatin structure leading todown-regulation of frataxin gene transcription.

Numerous data have demonstrated that analysis of GAA repeats constitutean essential part in the diagnosis of FA along with clinical diagnosis.Molecular genetic tests are also performed to identify carriers and inprenatal testing. Current FA diagnostic methods involve polymerase chainreaction (PCR) analysis and Southern blotting technique. The PCR test isperformed by amplification of the GAA repeat-containing DNA region inthe frataxin gene. The different PCR reactions that have been employedto map GAA repeat expansions are classical PCR, long-range PCR ortriplet-primed PCR (TP-PCR). In all cases, the size of the PCR fragmentis analyzed using agarose-gel electrophoresis and DNA sequencing. Inmost cases, both PCR and Southern blot are combined to complement theresults.

Problems encountered during amplification of medium and long sized GAArepeats (number of repeats >200) using PCR have been reported. Therepetitive nature of the expanded sequence and its ability to adoptH-DNA and higher order DNA structures are the two main factors causingpolymerase pausing leading to false results.

Thus, there is still a need in the art for alternative or improvedmethods for detecting the expanded GAA repeats to be used in thediagnosis of Friedreich's ataxia. Also, there is a need for therapies inorder to treat and/prevent Friedreich's ataxia.

SUMMARY OF INVENTION

The object of the present invention is to overcome or at least mitigatesome of the problems associated with the prior art.

This object is in once aspect obtained by the provision of anoligonucleotide comprising a sequence selected from the group consistingof (GAA)_(n), (CTT)_(n) or (JTT)_(n), wherein the oligonucleotide isbased on a peptide nucleic acid oligonucleotide or an equivalentoligonucleotide analogue, such as morpholino oligonucleotide or a lockednucleic acid oligonucleotide. The invention is also directed to apharmaceutical composition comprising such an oligonucleotide,optionally in combination with pharmaceutically acceptable carriers,adjuvants and/or excipients.

The oligonucleotides of the present invention were demonstrated to beable to dissociate and/or abolish the presence of H-DNA/higher order DNAstructures at GAA repeat sequences. Such GAA repeats are e.g. involvedin Friedreich's ataxia. This finding has large implications in thediagnosis and/or treatment of Friedreich's ataxia. Without wishing to bebound by theory, the oligonucleotides' ability to invade higher orderDNA structures, such as such structures in genomic DNA, is believed tobe a mechanism of importance for all aspects of the present document.

The invention is therefore directed to the use of the (GAA)_(n),(CTT)_(n) and/or (JTT)_(n) oligonucleotide or the pharmaceuticalcomposition for dissociating and/or abolishing the presence of higherorder DNA structures, such as H-DNA, at GAA repeat expansions and/or theuse of the (GAA)_(n), (CTT)_(n) and/or (JTT)_(n) oligonucleotide fordetermining the length and/or number of GAA repeats in a repeated GAAsequence, such as in the frataxin gene.

The present invention is in a further aspect directed to the (GAA)_(n),(CTT)_(n), and/or (JTT)_(n) oligonucleotide for use as a medicament.

The invention is also directed to the (GAA)_(n), (CTT)_(n), and/or(JTT)_(n) oligonucleotide or the pharmaceutical composition for use inthe prevention and/or treatment of Friedreich's ataxia. Also encompassedis the use of the (GAA)_(n), (CTT)_(n), and/or (JTT)_(n) oligonucleotideor the pharmaceutical composition for the preparation of a medicamentfor the prevention and/or treatment of Friedreich's ataxia. Further, theinvention is directed to a method for diagnosing Friedreich's ataxia ina subject comprising the steps of:

-   -   a) isolating DNA from a biological sample    -   b) optionally cleaving the DNA isolated in step a) with one or        more DNA restriction enzyme(s)    -   c) adding an oligonucleotide as defined herein to the isolated        DNA of step a) or the cleaved DNA of step b)    -   d) determining the length, sequence and/or number of GAA repeats        in the frataxin gene.        As the oligonucleotides of the invention have the ability to        affect and/or dissociate higher order structures of DNA at GAA        repeat sequences, the use of such oligonucleotides may enable a        more efficient and/or accurate determination of the number of        repeats in a GAA repeat sequence. Also, the dissociation and/or        abolishment of such higher order structures may enable a correct        functioning and/or expression of the frataxin gene.

DEFINITIONS

The oligonucleotides defined herein and used in different aspects ofthis document are based on oligonucleotide analogues. By “based on” ismeant that the oligonucleotides are built up by analogues of deoxyribo-or ribonucleotides including modifications of base, sugar and/orphosphodiester backbone. The oligonucleotide analogues useful in thepresent context are disclosed in more detail elsewhere herein.

“Peptide nucleic acid” (PNA) are DNA mimics containing a peptide likebackbone. PNA oligonucleotides are able to invade DNA structures andreplace DNA-DNA Watson-Crick hydrogen bonds in base pairs with new ones(PNA-DNA hydrogen bonds). This feature constitutes a key component ofthe present invention. PNA binds strongly to sequence complementary DNAor RNA with high specificity. PNA was originally designed to bind in asequence specific manner to the major groove of a DNA duplex forming anintermolecular triplex structure; however, it was soon discovered thatseveral other PNA-DNA complexes are also formed (9), (FIG. 2). Bindingof short homopyrimidine PNA to double strand DNA leads mainly toformation of a triplex invasion structure (FIG. 2C), which ispractically irreversible. The formation of a triplex invasion complex isslow and negatively affected by duplex DNA stabilizing conditions, suchas physiological salt concentrations. The use of bisPNA (bisPNA includetwo PNA oligonucleotides connected together through a flexible chemicallinker) increases PNA binding affinity of target DNA and kinetics.Negative supercoiling, and other processes that enhance stranddisplacement such as transcription increase the rate of triplex invasionby PNA. Longer PNA can form an intermolecular triplex structure (FIG.2B) at high salt concentrations, which forms predominantly with PNAbinding in the parallel direction (10). Intermolecular triplex andtriplex invasion PNA-DNA complexes have not been detected for homopurinePNAs. On the other hand, homopurine PNAs are able to form a duplexinvasion complex with double strand DNA (FIG. 2D).

A “locked nucleic acid” (LNA) are oligonucleotide analogues that have anability to invade DNA structures in a similar way as PNA. LNA are oftenreferred to as inaccessible RNA, and are modified RNA nucleotides. Theribose moiety of an LNA nucleotide is modified with an extra bridgeconnecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose inthe 3′-endo (North) conformation, which is often found in the A-formduplexes. LNA nucleotides can be mixed with DNA or RNA residues in theoligonucleotide whenever desired. The locked ribose conformationenhances base stacking and backbone pre-organization. This significantlyincreases the hybridization properties (melting temperature) ofoligonucleotides.

Morpholino oligonucleotides may invade DNA structures in an equivalentway as PNA and LNA. They are nucleic acids mimics where the deoxyribosesugar of DNA is replaced by a morpholine and the phosphodiester backboneby a phosphoroamidate linkage. Morpholino oligonucleotides have anuncharged backbone, are resistant to enzymatic digestion and are used totarget double strand DNA and RNA in a sequence-specific manner.

Oligonucleotides having a “natural” sugar and/or phosphodiester backbonewith or without base modifications are not able to invade DNA structuresnor dissociate higher order DNA structures.

H-DNA is an intramolecular DNA triplex structure that forms atpolypurine/polypyrimidine stretches with mirror-symmetry. H-DNA forms bythe disruption of the double helix DNA structure and folding back of oneof the single strands forming new hydrogen bonds with the duplex. Thisresults in the formation of a triplex and a single strand region (FIG.1B).

Higher order DNA structures refer to DNA structures formed by one orseveral non-B-DNA structures, including triplex DNA (H-DNA). An exampleof higher order DNA structures is sticky DNA, which may form at expandedGAA repeats in the first intron of the frataxin gene. Sticky DNA mayform as one long intramolecular triplex (H-DNA) or by the association oftwo or more triplex structures.

GAA repeat sequences refer to a double stranded DNA with a repeated GAAsequence on one strand and a complementary repeated CTT sequence on theother strand, such as in the frataxin gene. The number of GAA repeats inthe human frataxin gene may go up to 1700 repeats. Such repeated DNAsequences are in the context of the present document called GAA repeatsequences.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. A) Structure of BQQ and BQQ-OP. B) Purine and pyrimidine H-DNAmotif formed at GAA repeats. C) PNA and TFO sequences.

FIG. 2. DNA and DNA-PNA structures, thin line: polypurine strand, thickline: polypyrimidine strand, grey line: PNA. A) H-DNA as one example ofpossible intramolecular triplex structures. B) Intermolecular triplexstructure. C) Triplex invasion complex. D) Duplex invasion complex.

FIG. 3. Triplex-specific cleavage of linearized pMP179 by BQQ-OP. A)Schematic presentation of TFO directed triplex formation and the twofragments generated after BQQ-OP cleavage indicated as X (3814 bp) and Y(3178 bp). B) Linearized pMP179 (Apa1) were incubated with 10 μM PNA3320, PNA 3482 or 4 μM CTT-TFO in cacodylate buffer at pH 6.5 with 100mM NaCl and with or without 2 mM MgCl₂ as indicated in the figure.Following, the plasmid was cleaved by BQQ-OP (1.5 μM) in the presence ofCu²⁺ and MPA (lanes 1-6) generating two fragments of the approximatesize 3814 and 3178 bp. Linearized pMP179 (Lin) is also shown.

FIG. 4. Triplex-specific cleavage of pMP179 by BQQ-OP. A) Schematicpresentation of pMP179. The two fragments generated from BQQ-OP cleavagefollowed by enzymatic digestion are indicated as X (3814 bp) and Y (3178bp). B) The plasmid was incubated with 10 μM of PNA 3320 or PNA 3482 inbuffer (lane 1 and 2) (sodium cacodylate 10 mM, pH 7.5 and 100 mM NaCland 2 mM MgCl₂). As reference the plasmid was also incubated with thesame concentration of CTT and GAA TFO (lane 4 and 5) or in the absenceof any oligonucleotide (lane 3). The plasmid was then cleaved by BQQ-OP(1.0 μM) in the presence of Cu²⁺ and MPA. Following, the plasmid wasdigested by ApaI generating two fragments of the approximate size 3814and 3178 bp. Supercoiled (SC) and linearized (Lin) pMP 179 and amolecular weight DNA ladder (M) are also included as references.

FIG. 5. A) DNA chemical modification pMP141 containing 9 GAA repeats.The plasmid was incubated in buffer (sodium cacodylate 10 mM, pH 7.5 and100 mM NaCl and 2 mM MgCl₂) in the absence (−) or in the presence of 10μM of PNA 3482 (CTT-PNA) or PNA 3320 (GAA-PNA). The plasmid was thentreated by 2% CAA (lane CAA)_(n) for 30 min. All samples were linearizedby ApaI before used as template for the primer reaction. As a controlthe plasmid was incubated at similar condition without addition of CAA.Linearized plasmid and sequencing reaction using dideoxynucleotides areused as references. B) Models showing possible structures formed at thepMP 141 plasmid. From the top, H-DNA formed in the absence of PNA,triplex invasion formed by PNA 3482, duplex invasion formed by PNA 3320.Nucleotides modified by CAA are indicated (▴).

FIG. 6. A) Affinity cleavage of pMP141 containing 9 GAA repeats. Theplasmid was incubated in buffer (sodium cacodylate 10 mM, pH 7.5 and 100mM NaCl and 2 mM MgCl₂) in the absence (−) or in the presence of 10 μMof PNA 3320 (GAA-PNA) or PNA 3482 (CTT-PNA). The plasmid was thencleaved by 1 μM BQQ-OP for 3 hours in 37° C. All samples were linearizedby ApaI before used as template for the primer extension reaction.Linearized plasmid and sequencing reaction using dideoxynucleotides areused as references. B) Models showing possible structures formed at thepMP141 plasmid. From the top, H-DNA formed in the absence of PNA,triplex invasion formed by PNA 3482, duplex invasion formed by PNA 3320.Nucleotides cleaved by OP are indicated (▴).

FIG. 7. A) Affinity cleavage and DNA chemical modification of pMP178containing 75 GAA repeats. The plasmid was incubated in buffer (sodiumcacodylate 10 mM, pH 7.5 and 100 mM NaCl and 2 mM MgCl₂) in the absence(−) or in the presence of 10 μM of PNA 3320 (GAA-PNA) or PNA 3482(CTT-PNA). The plasmid was then cleaved by 2% CAA (lanes CAA)_(n) for 30min or by 1 μM BQQ-OP (lanes BQQ-OP) for 3 hours at 37° C. All sampleswere linearized by ApaI before used as template for the primer extensionreaction. As a control the plasmid was incubated at similar conditionwithout addition of a cleavage reagent. Linearized plasmid andsequencing reaction using dideoxynucleotides are used as references. B)Models showing possible structures formed at pMP 179. Thin line=purinestrand, thick line=pyrimidine strand, grey line ═PNA. 1) H-DNA. 2) H-DNAtrapped by PNA invasion complex. 3) Invasion complex. 4) Triplexstructure formed by PNA as the third strand. 5) Duplex invasion. Regionsmodified by CAA (▴) or cleaved by BQQ-OP (o) are indicated.

DETAILED DESCRIPTION OF INVENTION

The present invention is based on the surprising finding that peptidenucleic acid (PNA) oligonucleotides are able to effect and/or dissociatea higher order DNA structure, including H-DNA, at expanded GAA repeats,and the abolishment of the interfering DNA structures in theamplification and detection of GAA repeats. The use of a (GAA)₄ (SEQ IDNO 4) PNA has for example been shown to completely abolish the presenceof higher order structure in vitro, due to its ability to invade DNAstructures. Alternatively, oligonucleotides equivalent to PNA in theirability to invade DNA structures, such as a morpholino or a lockednucleic acid may be used in the present invention. This finding enabledthe development of new techniques for detecting expanded GAA repeats,which e.g. may be used in the diagnosis of Friedreich's ataxia. Also,this finding enabled new methods and compositions for preventing and/ortreating Friedreichs's ataxia.

The oligonucleotide analogues in the invention target the GAA repeats onthe DNA level to dissociate and/or abolish the higher order DNAstructures formed at these repeats, in contrast to reports elsewhere(11) where oligonucleotides are used to target RNA. The oligonucleotideanalogues in the invention target the inherent higher order DNAstructures at the frataxin gene and not any other structure that mayform during transcription (12). Without wishing to be bound by theory,for all aspects of the invention, the ability of the oligonucleotideanalogues to invade DNA structures at GAA repeats is an importantfeature leading to the dissociation and/or abolishment of DNA higherorder structures at GAA repeats within a gene (such as in genomic DNA).

The invention is in one aspect directed to an oligonucleotide comprisinga sequence selected from the group consisting of (GAA)_(n) (SEQ ID NO1), (CTT)_(n) (SEQ ID NO 2) or (JTT)_(n) (SEQ ID NO 2) (or a sequence(CTT/JTT)_(n) comprising a mixture of CTT and JTT triplets in any numberand order as long as the total number does not exceed n) wherein theoligonucleotide is based on a peptide nucleic acid oligonucleotide or anequivalent oligonucleotide analogue, such as morpholino or a lockednucleic acid.

It is in the context of the present invention to be understood that theoligonucleotide comprises n numbers of the GAA, CTT or JTT triplets.However, an oligonucleotide can comprise of a mixed sequence of nnumbers of CTT and JTT triplets, as also is evident from SEQ ID NO 2. Inall aspects of the invention, typically only one of the (GAA)_(n),(CTT)_(n) or (JTT)_(n) oligonucleotides (or a mixed (CTT/JTT)_(n)oligonucleotide) will be used. However, a mixture of the different kindsof oligonucleotides may be used in the different aspects of theinvention.

The oligonucleotide for all aspects of the invention comprises asequence selected from (GAA)_(n), (CTT)_(n) or (JTT)_(n) or a sequencecomprising a mixture of JTT and CTT. When the oligonucleotide is a mixed(CTT/JTT)_(n) oligonucleotide, any number and order of CTT and JTTtriplets, respectively, is encompassed, as long as the total number ofrepeats and/or residues is in conformity with the information givenelsewhere herein. Such a mixed (CTT/JTT)_(n) sequence is also shown inSEQ ID NO 2. The oligonucleotide is always based on peptide nucleic acidor an equivalent oligonucleotide analogue, such as morpholino or alocked nucleic acid, even if not always explicitly mentioned. Forexample, the oligonucleotide may be (GAA)_(n) based on peptide nucleicacid, morpholino or a locked nucleic acid, such as (GAA)_(n) based onpeptide nucleic acid. It may also be (CTT)_(n) based on peptide nucleicacid, morpholino or a locked nucleic acid. Further it may be (JTT)_(n)based on peptide nucleic acid, morpholino or a locked nucleic acid.Also, it may be a mixed (CTT/JTT)_(n) oligonucleotide based on peptidenucleic acid, morpholino or a locked nucleic acid. The nucleotide “J”stands for pseudoisocytosine. Typically, the oligonucleotide is based onPNA, even if an oligonucleotide based on morpholino or a locked nucleicacid also may be used. For all aspects of the present document, theoligonucleotide may consist of a sequence selected from the groupconsisting of (GAA)_(n), (CTT)_(n) or (JTT)_(n) or a mixed(CTT/JTT)_(n). The invention is also directed to a pharmaceuticalcomposition comprising such an oligonucleotide.

The (GAA)_(n), (CTT)_(n) or (JTT)_(n) oligonucleotide, when used todissociate a higher order DNA structure (such as in genomic DNA) indifferent aspects of this document, may further comprise a terminalflanking sequence in one or both ends of said oligonucleotide. Flankingsequences are used herein to facilitate dissociation of theoligonucleotide of the invention in a DNA polymerase reaction. Thismeans that this terminal flanking sequence may be present at the N-and/or C-terminal of an oligonucleotide based on PNA, or in the 5′and/or 3′ end of an oligonucleotide based on a morpholino or lockednucleic acid. This flanking sequence is substantially non-complementaryto a GAA or CTT repeat sequence. By “substantially non-complementary” ismeant that the sequence has a hybridization Tm (melting temperature)that is ≦37° C. Melting temperature is the temperature at which 50% ofthe oligonucleotide and its perfect sequence complement are in duplex.The length of the flanking sequence is typically about 2-10 nucleotides,such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. Exemplary flankingsequence of the present invention is TGCACGCGCT. The flanking sequencemay be based on the same kind of oligonucleotide as the oligonucleotideitself, e.g. a PNA oligonucleotide may have a PNA flanking sequence, butmay just as well have a flanking sequence based on e.g. DNA. Anoligonucleotide for use in the present invention may therefore consistof a (GAA)_(n), (CTT)_(n) or (JTT)_(n) oligonucleotide linked to aflanking sequence as defined herein. The invention is therefore alsodirected to a nucleotide sequence comprising or consisting of such a(GAA)_(n), (CTT)_(n) or (JTT)_(n) oligonucleotide and a flankingsequence. As further discussed elsewhere in this document, when anoligonucleotide is labeled with a label for detection purposes, thisflanking sequence is not to be present.

In all aspects of the invention, the number of repeats, “n”, in theoligonucleotide is typically about 2-10, such as 2, 3, 4, 5, 6, 7, 8, 9or 10. The number of repeats, n, need not to be an integer, i.e. thefirst and/or last repeat may be constituted by only one or two residuesof the triplet repeat sequence. The number of residues in theoligonucleotide is typically about 6-30, such as 12-21. The number ofresidues may therefore be e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. Exemplaryoligonucleotides of the present invention areAc-TTCTTCTTCTTCTTC-eg1-Lys-NH₂ (i.e. SEQ ID NO 3 with a N-terminal Acand a C-terminal eg1-Lys-NH₂, eg 1 denoting an ethylene glycol linker),H-LysLys-GAAGAAGAAGAA-Lys-NH₂ (i.e. SEQ ID NO 4 with an N-terminalH-LysLys and a C-terminal Lys-NH₂), Ac-TTCTTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂(i.e. SEQ ID NO 5 with N-terminal Ac and C-terminal eg1),Acr-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂ (i.e. SEQ ID NO 3 withN-terminal Acr-(diMeLys)₂ and C-terminal eg1-Lys-NH₂)Ac-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂ (i.e. SEQ ID NO 3 withN-terminal Ac-(diMeLys)₂ and C-terminal eg1-Lys-NH₂),Ac-TTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂ (i.e. SEQ ID NO 7 with N-terminal Acand C-terminal eg1-Lys-NH₂), H-LysLys-GAAGAAGAAGAAGAA-Lys-NH₂ (i.e. SEQID NO 8 with N-terminal H-LysLys and C-terminal Lys-NH₂),H-LysLys-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂ (i.e. SEQ ID NO 6 with N-terminalH-LysLys and C-terminal Lys-NH₂), Acr-eg1-GAAGAAGAAGAA-Lys-NH₂ (i.e. SEQID NO 4 with an N-terminal Ac-eg1 and a C-terminal Lys-NH₂)Acr-eg1-GAAGAAGAAGAAGAA-Lys-NH₂ (i.e. SEQ ID NO 8 with N-terminalAcr-eg1 and C-terminal Lys-NH₂), Acr-eg1-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂(i.e. SEQ ID NO 6 with N-terminal Acr-eg1 and C-terminal Lys-NH₂) allpreferably being based on PNA. The present N- and C-terminal chemicalgroups are eg1=ethylene glycol linker, Lys=Lysine, Ac=acetyl,Acr=acridine, diMeLys=dimethyl lysine. The N- and C-terminal sequencesmay consist of other chemical groups. Further, the N- and C-terminalgroups exemplified above may also be used for other oligonucleotidesencompassed by this document. The sequences may also be used withouttheir N- and/or C-terminal chemical groups.

As a (GAA)_(n), (CTT)_(n) or (JTT)_(n) oligonucleotide (or mixed(CTT/JTT)_(n) oligonucleotide as discussed elsewhere herein) as definedherein may be used to dissociate higher order structures in a GAA repeatsequence, such an oligonucleotide may be used as a medicament, e.g. forthe prevention and/or treatment of Friedreich's ataxia. Although notwishing to be bound by theory, it is speculated that the dissociation ofthe higher order structure, such a triplex (H-DNA) formations, againenables wild type replication and/or transcription of the frataxin gene.The invention is therefore also directed to such an oligonucleotide foruse as a medicament. The invention is further directed to such anoligonucleotide for use in the prevention and/or treatment ofFriedreich's ataxia. In addition, the invention is directed to the useof such an oligonucleotide for the preparation of a medicament for theprevention and/or treatment of Friedreich's ataxia. Alternatively, apharmaceutical composition as defined herein may be used for thesepurposes. The invention is also directed to the use of a (GAA)_(n),(CTT)_(n) or (JTT)_(n) oligonucleotide as defined herein as apharmaceutical.

When the oligonucleotides disclosed herein are used as a pharmaceutical,they invade higher order structures formed at GAA repeats in genomic DNAand act to treat and/or prevent Friedreich's ataxia by dissociatingand/or abolishing these higher order DNA structures in the genomic DNAof the subject to which the pharmaceutical is administered. An advantagewith the present invention is that the oligonucleotides of the inventionare resistant to enzymatic digestion and have a long half-life incellular environment. Also, the oligonucleotide-DNA complexes, such asPNA-DNA, are very stable. Taken together, less frequent administrationof the oligonucleotide may be used when the oligonucleotides is used forthe prevention and/or treatment of Friedreich's ataxia. The highstability of the oligonucleotide-DNA complexes is also advantageous whenthe oligonucleotides are used for diagnosis of Friedreich's ataxia.

A pharmaceutical composition comprising the oligonucleotide of theinvention may also optionally comprise one or more of a pharmaceuticallyacceptable carrier, adjuvant and/or excipient, as is commonly known inthe art in this kind of pharmaceutical compositions. The oligonucleotidemay e.g. be present in a physiologically acceptable salt solution orbuffer. In a pharmaceutical composition in accordance with the presentinvention, conjugation of the oligonucleotide to one of many well-knowncarriers such as nuclear localization signal peptides (NLS), cationic orcell penetrating peptides (CPP) or use of uptake enhancers, such aslipophilic compounds that improve cell delivery or pharmacodynamicproperties, may be used. Excipients may be used to improve solubility,stability and uptake. The pH of the pharmaceutical composition isselected so as to be biologically compatible. The concentration of anoligonucleotide of the invention in such a pharmaceutical composition istypically about 5-50 mg/ml. The amount administered to a subject istypically 0.1-6 mg/kg.

Also encompassed is a method for treating Friedreich's ataxia comprisingthe step of administering a therapeutically effective amount of anoligonucleotide as defined herein or a pharmaceutical composition asdefined herein to a subject in need of such treatment. The mechanism forsuch a treatment most likely takes place by the oligonucleotide(s)invading the genomic DNA thereby dissociating and/or abolishing higherorder DNA structures at GAA repeats in genomic DNA. The administrationmay take place e.g. by intravenous or local administration, such as byintrathecal administration.

As the oligonucleotides disclosed herein may be used for dissociatinghigher order DNA structures, the invention is also directed to the useof an oligonucleotide as defined herein for affecting, dissociatingand/or abolishing the formation of higher order DNA structures, such astriplex (H-DNA) formations, at GAA repeat expansions. The document istherefore directed also to the use of a (GAA)_(n), (CTT)_(n) and/or(JTT)_(n) oligonucleotide (or mixed (CTT/JTT)_(n) oligonucleotide) or apharmaceutical composition as defined herein for dissociating and/orabolishing the formation of higher order DNA structures, such as triplexformation, at GAA repeats.

Such an effect may take place in vitro or in vivo. The GAA repeats maye.g. be present in genomic DNA.

The use of the oligonucleotides as defined herein fordissolving/abolishing higher order DNA structures, also enables the useof such oligonucleotides for determining the length and/or number of GAArepeats in a GAA repeat sequence, such as in the frataxin gene. Thepresent document is therefore also directed to the use of a (GAA)_(n),(CTT)_(n) and/or (JTT)_(n) oligonucleotide (or mixed (CTT/JTT)_(n)oligonucleotide) for determining the length and/or number of GAA repeatsin a repeated GAA sequence, such as in the frataxin gene.

The uses of the oligonucleotides for dissociating and/or abolishinghigher order DNA structures is believed to take place by theoligonucleotides invading higher order DNA structures formed at GAArepeats in DNA, such as genomic DNA, thereby dissociating and/orabolishing such structures.

This document is therefore also directed to a method for diagnosingFriedreich's ataxia in a subject comprising the steps of:

a) isolating DNA from a biological sample;b) optionally cleaving the DNA isolated in step a) with one or more DNArestriction enzyme(s)c) adding an oligonucleotide, said oligonucleotide comprising a sequenceselected from the group consisting of (GAA)_(n), (CTT)_(n), (JTT)_(n)(or a mixed (JTT/CTT)_(n) oligonucleotide as discussed elsewhere herein)wherein said oligonucleotide is based on a peptide nucleic acidoligonucleotide or an equivalent oligonucleotide analogue, such asmorpholino or a locked nucleic acid, to the isolated DNA of step a) orthe cleaved DNA of step b)d) determining the length, sequence and/or number of GAA repeats in thefrataxin gene.

The biological sample from which DNA is isolated in step a) is typicallya sample containing cells. Typically such a sample is a blood sample,but it may also be a tissue sample. Typically the sample containsgenomic DNA. Also, the above disclosed method may be used for researchpurposes wherein the sample e.g. may be cell culture sample or microbialsample (such as a fungal or bacterial sample) containing a GAA repeatsequence. Typically RNA and proteins are removed by RNase treatment andprecipitation by standard methods known to the person skilled in theart. The above disclosed method may therefore also be used fordetermining the length, sequence and/or number of GAA repeats in a GAArepeat sequence more generally in any kind of sample potentiallycontaining such a GAA repeat sequence and not only for diagnosingFriedreich's ataxia. The GAA repeat sequence is typically a GAA repeatsequence from the frataxin gene, but other GAA repeat sequencespotentially forming higher order structures may also be analyzed by themethods of the present document.

In all aspects of the present document, the target for theoligonucleotides disclosed herein is typically genomic DNA.

The isolation of DNA from the sample in step a) in the above method maytake place by any commonly known method for isolating DNA. The skilledperson knows which method to employ depending on the origin of thesample containing the DNA. For example, commercially available kit forDNA isolation may be used, e.g. the “DNA Isolation Kit for Cells andTissues” or “DNA Isolation Kit for Mammalian Blood” from Roche AppliedSciences, or the “QTAamp DNA Blood Mini Kit” from Qiagen.

In step b) of the above disclosed method, the DNA is optionally cleavedwith one or more DNA restriction enzyme(s) in order to facilitate theanalysis of the sample. The cleavage of the DNA may aid in thesubsequent analysis. In Southern blot, cleavage of genomic DNA isnecessary to enable analysis through gel electrophoresis. For PCR andsequencing, cleavage of genomic DNA is optional and may helpdissociation of higher order DNA structures. However, this step is onlyoptional. Examples of suitable DNA restriction enzymes include, but arenot limited to, BsiHKAI.

To the isolated DNA obtained in step a) or the cleaved DNA of step b),an oligonucleotide (as defined elsewhere in this document as regards thenumber of repeats (n), residues, flanking sequences and preferredsequences) is then added. In particular the oligonucleotide may be(GAA)_(n) based on peptide nucleic acid. The addition of thisoligonucleotide dissociates/abolishes the higher order structure,including triplex/H-DNA of a GAA repeat sequence present in the sampleand will therefore affect a higher order DNA structure in the frataxingene. The added oligonucleotide invades the DNA structures andhybridizes with the GAA repeat sequence and forms an oligonucleotide-DNAcomplex within the GAA repeat sequence. However, this binding does notinterfere with the analysis methods for determining the length, sequenceand/or number of GAA repeats of the GAA repeat sequence. Thus,dissociation/abolishment of the DNA higher order structures at GAArepeat sequences is facilitated, as the higher order DNA structuresinterfere with the methods commonly used for anal yzing the length,sequence and/or number of GAA repeats of a GAA repeat sequence. Thesurprising finding by the present inventors that a (GAA)_(n), (CTT)_(n)or (JTT)_(n) oligonucleotide based on PNA, morpholino or locked nucleicacid, with or without a flanking sequence as disclosed herein candissociate and/or abolish higher order DNA structures therefore enablesa much more efficient and/or accurate analysis of the length, sequenceand/or number of GAA repeats in a GAA repeat sequence, such as in thefrataxin gene. The use of the flanking sequence has one effect ofminimizing the risk that the oligonucleotide functions as a clamp assuch an oligonucleotide clamp may prevent elongation by polymerase.Also, the use of a flanking sequence may enable the enzyme to displacethe oligonucleotide and read through the DNA sequence.

Before addition of the oligonucleotide in step c) the oligonucleotide istypically heated at a temperature of about 37-95° C. for typically about1-10 mM. The oligonucleotide is then incubated with the isolated DNA ofstep a) or the cleaved DNA of step b), typically at a temperature ofabout 4-50° C. for a time period typically of about 10-120 min. Theoligonucleotide is typically present in an aqueous solution, such aswater. Typically, a large excess of oligonucleotide to DNA is added,such as 500-10000 times more oligonucleotide (on molar basis). Thehybridization typically takes place in water or salt buffer, at a pH ofabout 6-9, for example 10 mM Tris-HCl or 10 mM sodium cacodylate or 140mM KCl. Without wishing to be bound by theory, the oligonucleotide(s)added invades GAA repeat sequences in the DNA sample, binds to acomplementary DNA sequence and thereby dissociates and/or abolish thepresence of higher order DNA structures at the specific genomic region.

In step d) a standard method for determining the length, sequence and/ornumber of GAA repeats may be used, such as polymerase chain reaction(PCR), primer extension reaction (PE), DNA sequencing or Southernblotting. The number of GAA repeats in the (genomic) DNA analyzed isthen compared to the number of GAA repeats in a healthy individual(generally not more than 40 repeats) in order to determine whether ornot the subject is afflicted with or at risk for developing Friedreich'sataxia and to determine the exact length of the disease-related GAArepeat.

There are three major steps in a PCR reaction. These steps are generallyrepeated for 20 or 40 cycles. The first step is the denaturation. Duringthe denaturation, the double strand melts open to single stranded DNA,all enzymatic reactions stop. The second step is the annealing whereinthe primers are allowed to bind to the template, and the polymerase canattach and starts copying the template. The third step is the extension,wherein the bases (complementary to the template) are coupled to theprimer on the 3′ side by the action of a polymerase enzyme (thepolymerase adds dNTP's from 5′ to 3′, reading the template from 3′ to 5′side, bases are added complementary to the template).

If PCR is used in step d) for determining the length, sequence and/ornumber of GAA repeats, a common PCR reaction is run. Primerscomplementary to the genomic regions (20-1500 nucleotides in 3′ and 5′flanking regions of the GAA repeat in intron one of the frataxin gene)and/or within the GAA repeat in intron one of the frataxin gene aretypically used for the PCR reaction. The primers are typically about10-50 nucleotides long. Typically in such a PCR reaction, the initialDNA denaturing step lasts for about 1-20 min and the denaturing step ineach PCR cycle for about 1-20 min.

If PE is used in step d) for determining the length, sequence and/ornumber of GAA repeats, a common PE reaction is run using genomic DNAfrom step b). A primer complementary to either of the genomic regions(20-1500 nucleotides in 3′ and 5′ flanking regions of the GAA repeat inintron one of the frataxin gene) and/or within the GAA repeat in intronone of the frataxin gene are typically used for the PE reaction. Theprimers are typically about 10-50 nucleotides long. Typically in such aPE reaction, the initial DNA denaturing step lasts for about 1-20 minand the denaturing step in each PE cycle for about 1-20 min.

The PCR or PE reaction product obtained after the PCR or PE reaction maybe hybridized to a labeled oligonucleotide comprising a sequenceselected from (GAA)_(n), (CTT)_(n) or (JTT)_(n) based on PNA, morpholinoor locked nucleic acid as disclosed elsewhere herein. Typically,oligonucleotides without the flanking sequence is used for thishybridization. Also, when used for hybridization purposes, theoligonucleotide is labelled with common label for detectinghybridization, such as a fluorescing label (e.g. Fluorescein, Bodipy,Texas red or cyanine dyes, e.g. Cy2, Cy3 and Cy5), a radioactive labeland/or biotin. Such labels are known to the person skilled in the art.It is to be understood that in the context of the present invention,such a labeled oligonucleotide is only to be used for detectionpurposes, i.e. an oligonucleotide used for affecting and/or dissolving ahigher order structure, such as in step c) of the method for diagnosingFriedreich's ataxia, or when the oligonucleotide is used in apharmaceutical composition or in the prevention and/or treatment ofFriedreich's ataxia, is not labeled with a label. The labeledoligonucleotide binds to any complementary PCR or PE reaction productwhich presence can be visualized with the aid of the label. It ispreferred to use a labeled oligonucleotide based on PNA, morpholino orlocked nucleic acid, as such oligonucleotides have a higher affinity forsingle and double strand DNA and an ability to invade DNA structures, ascompared to a DNA-based oligonucleotide, thereby e.g. facilitatingdetection of hybridized sequences. The hybridization of the labeledoligonucleotide with the PCR or PE reaction product is typicallyperformed by incubating the labeled oligonucleotide with the PCR or PEreaction product at about 4-50° C. for about 10-120 min. The labeledoligonucleotides may optionally be heated before the hybridization atabout 37-95° C. for about 1-10 min. The hybridization to labeledoligonucleotide typically takes place in water or salt buffer at a pH ofabout 6-9, for example at buffer containing 10 mM Tris-HCl, pH 8.3, 50mM KCl, 1.5 mM MgCl₂, 0.001 gelatin or a buffer containing 75 mMTris-HCl pH 8.8, 20 mM (NH₄)₂SO₄, 0.01% (v/v) Tween 20. In thishybridization, typically, a large excess of oligonucleotide to DNA isadded, such as 500-10000 times more oligonucleotide (on molar basis).

The PCR or PE reaction product, optionally hybridized to the labeledoligonucleotide, is thereafter run on a gel, such as an agarose gel. Theperson skilled in the art is well acquainted with how to prepare and runsuch gels. In one lane on the gel, a DNA ladder marker is run, therebyenabling the determination of the size of PCR or PE reaction products.Such DNA ladder markers commonly comprises a mixture of nucleic acidfragments of different sizes and consequently of different weights, thusenabling the determination of the size of other nucleic acid fragmentsrun on the gel by comparing their size with the DNA ladder marker.Ethidium bromide staining of nucleic acids on the gel is typically usedto visualize nucleic acid, including the DNA ladder marker. However, anyPCR or PE reaction product to which the labeled oligonucleotide hashybridized may also be visualized via the label. Also, binding oflabeled oligonucleotide to the PCR or PE reaction product offers asequence-specific detection of the GAA repeat-containing fragmentwhereas standard staining of gel (e.g. ethidium bromide or cybergreen)detect DNA fragments at all different sequences. By comparing the sizeof the PCR or PE reaction product hybridized to the labeledoligonucleotide to the DNA ladder marker, the size and/or the number ofrepeats of the PCR or PE reaction product may be determined.

Step d) of the above disclosed method may also be performed bysequencing the frataxin gene in the sample of step c). Any commonly usedmethod for sequencing DNA may be used. A sequencing reaction maygenerally be performed in the following manner. There are three majorsteps in a sequencing reaction which are repeated for 30 or 40 cycles.Firstly, the sample is denatured. During the DNA denaturing step, thedouble strand melts open to single stranded DNA, all enzymatic reactionsstop. Then a primer complementary to one strand is annealed (insequencing reactions, only one primer is used, so there is only onestrand copied). The extension reaction then takes place. The bases(complementary to the template) are coupled to the primer on the 3′ side(adding dNTP's or ddNTP's from 5′ to 3′, reading from the template from3′ to 5′ side, bases are added complementary to the template). When addNTP is incorporated, the extension reaction stops because in ddNTP ahydrogen has replaced the OH-group at the 3′-position. After thesequencing reactions, the mixture is separated by gel electrophoresis ona polyacrylamide gel. The sample may be detected on an automatedsequencer using fluorescently labeled nucleotides. Each base has its owncolor, so the sequencer can detect the order of the bases in thesequenced gene: The fluorescently labeled fragments that migrate throughthe gel are passing a laser beam at the bottom of the gel. The laserexcites the fluorescent molecule, and the wavelength is registered by aspectrophotometer.

Alternatively, step d) of the above disclosed method may be performed bySouthern blotting using the same labeled oligonucleotides as describedabove for analysis of the PCR or PE reaction product. A Southern blot istypically performed by digesting genomic DNA with one or morerestriction enzymes (corresponding to step b) above) and separating theresulting fragmented DNA on an agarose gel. The DNA is then typicallydenatured in a solution containing NaOH before transferring the DNA to anitrocellulose or nylon membrane by placing such a membrane on the geland the DNA transferred from the gel to the membrane by capillaryaction. The DNA is then baked in vacuum or a regular oven to fix the DNAto the membrane. Alternatively UV light immobilization may be used. Ahybridization probe is then allowed to hybridize to the membrane. In thecontext of the present invention, the probe is a labeled oligonucleotidecomprising a sequence selected from (GAA)_(n), (CTT)_(n) or (JTT)_(n)based on PNA, morpholino or locked nucleic acid as disclosed elsewhereherein. Typically, oligonucleotides without the flanking sequence isused for this hybridization. Also, when used for hybridization purposes,the oligonucleotide is labelled with common label for detecting ifhybridization has occurred, such as a fluorescing label, such asFluorescein, Bodipy or cyanine dyes, a radioactive label and/or biotin.Excess probe is then washed away and a detection method suitable for thelabel used is used to detect the size of the (expanded) GAA repeat.Typically, a large excess of oligonucleotide to DNA is added, such as500-10000 times more oligonucleotide (on molar basis).

The present document also discloses an in vitro method for diagnosingFriedreich's ataxia and/or determine the length, sequence and/or numberof GAA repeats in a frataxin gene or other GAA repeat sequence. Such amethod comprises the steps of i) adding an oligonucleotide to a DNAsample, said oligonucleotide comprising a sequence selected from thegroup consisting of (GAA)_(n), (CTT)_(n), (JTT)_(n) (or a mixed(JTT/CTT)_(n) oligonucleotide as discussed elsewhere herein) whereinsaid oligonucleotide is based on a peptide nucleic acid oligonucleotideor an equivalent oligonucleotide analogue, such as morpholino or alocked nucleic acid said step of adding an oligonucleotide to a DNAsample optionally being preceded by a step i′) of cleaving the DNAsample with one or more restriction enzyme(s) and ii) determining thelength, sequence and/or number of GAA repeats in the frataxin gene. Thesteps i), i′) and ii) of this in vitro method are performed as describedabove for the corresponding steps in the method for diagnosingFriedreich's ataxia in a subject. Step i) of the in vitro methodtherefore corresponds to step c) of the above method, step i′)corresponds to step b) and step ii) corresponds to step d).

The DNA sample analysed in the above two disclosed methods potentiallycomprises an expanded GAA repeat sequence.

EXPERIMENTAL SECTION

In the present invention, a model system based on a triplex-specificcleaving reaction based on a triplex intercalatingbenzoquinoquinoxaline-1,10-phenanthroline (BQQ-OP) compound (FIG. 1A)and a chemical modification reaction using chloroacetaldehyde (CAA) wasexamined. Both reactions were analyzed using a primer extension reactionand denaturing polyacrylamide and/or agarose gel electrophoresis. First,the ability of BQQ-OP to mediate a triplex-specific DNA double strandcleavage of PNA-DNA triplex structures in plasmid containingpathological GAA repeats was validated. Then, the BQQ-OP assay wasemployed to demonstrate the different binding modes of (GAA)_(n) and(CTT)_(n) PNA to intramolecular triplex (H-DNA) forming GAA repeats atone nucleotide resolution.

Example 1 Materials and Methods Plasmids:

GAA-containing plasmids, pMP179, pMP178 and pMP141 (115, 75 and 9repeats, respectively) were a kind gift from Prof M. Pandolfo'sLaboratory. The plasmids are derived from pSPL3 as described in theliterature and are all flanked by 352 and 256 by of human frataxingenomic sequences 5′ and 3′ of the GAA repeat, respectively (4).

Analysis of Triplex Motif Formation in Linear Plasmids in the Presenceof PNA:

Plasmid pMP 179 was linearized by ApaI, followed by DNA isolation usingminiprep column (Qiagen). 0.2 μg linearized pM179 was incubated (2.2 nMin total volume of 20 μl) at 37° C. for 1 h in the presence of a 12-mer(GAA) (FIG. 1C, PNA 3320) or a 15-mer (CTT) (FIG. 1C, PNA 3482) PNA (10μM), or a 20-mer (CTT)_(n) oligonucleotide (4 μM) (FIG. 1C) incacodylate buffer (10 mM, pH 6.5 and (100 mM NaCl and 0, or 2 mM MgCl₂).BQQ-OP (1.5 μM) and CuSO₄ (2 μM) were premixed at room temperature for15 min and then added to the plasmid. The mixture was left for 25 min atroom temperature and mercaptopropionic acid (MPA, 2 mM, final volume 20μl) was added to initiate the cleavage reaction that was allowed toproceed for 2 h at 37° C. The samples were then analyzed using 0.7%agarose gel electrophoresis (50 V, 1 h) and ethidium bromide staining.Gel-doc XR with Quantity One 4.5.2 software (Bio-Rad) was used for gelanalysis and quantification. MassRuler (Fermentas) was used as amolecular weight DNA ladder.

Analysis of Triplex Motif Formation in Supercoiled Plasmids in thePresence of PNA:

Plasmid pMP179 (1 μg) was incubated in buffer (sodium cacodylate 10 mM,pH 7.5 and 100 mM NaCl and 2 mM MgCl₂) at 37° C. for 2 h in the in thepresence of either a 12-mer (GAA) (PNA 3320), a 15-mer (CTT) PNA (PNA3482) (10 μM), a 20-mer (GAA)_(n) oligonucleotide (10 μM) or a 20-mer(CTT)_(n) oligonucleotide (10 μM) (FIG. 1C). BQQ-OP (1 μM) and CuSO₄(1.5 μM) were premixed at room temperature for 15 min and added to theplasmid solution. The mixture was left for 45 min at room temperatureand mercaptopropionic acid (MPA, 2 mM, final volume 20 μL) was added toinitiate the cleavage reaction. The reaction was allowed to proceed for3 h at 37° C., followed by isolation of the DNA using miniprep column(Qiagen). As a control, plasmid pMP 179 was cleaved in the absence ofPNA or oligonucleotides using BQQ-OP and similar experimental conditionsas above. The isolated DNA was digested using ApaI (1 U, Promega) forthree hours at 37° C. and then analyzed using 0.7% agarose gelelectrophoresis (50 V, 1 h) and ethidium bromide staining. Gel-doc XRwith Quantity One 4.5.2 software (Bio-Rad) was used for gel analysis andquantification. MassRuler (Fermentas) was used as a molecular weight DNAladder.

Affinity cleavage and chemical modification of DNA and primer extensionreaction 1 μg of plasmid pMP178 (11.2 nM) or pMP141 (11.5 nM) wasincubated in buffer (sodium cacodylate 10 mM, pH 7.5 and 100 mM NaCl (or140 mM KCl) and 2 mM MgCl₂) at 37° C. for 2 h in the absence or presenceof either the 12-mer PNA 3320 or the 15-mer PNA 3482 (20 μM) (FIG. 1C).

BQQ-OP Cleavage:

BQQ-OP (1 μM) and CuSO₄ (1.5 μM) were premixed at room temperature for15 min and added to the plasmid solution. The mixture was left for 45min at room temperature and mercaptopropionic acid (MPA, 2 mM, finalvolume 20 μL) was added to initiate the cleavage reaction. The reactionwas allowed to proceed for 3 h at 37° C., followed by isolation of theDNA using miniprep column (Qiagen). The isolated DNA was digested usingApaI (Fermentas Fastdigest) and then was the enzyme inactivated in 65°C. for 5 min.

Chloroacetaldehyde (CAA)_(n) chemical modification: CAA (2%) was addedto the plasmid solution (final volume 20 μL) and the reaction wasallowed to proceed in 37° C. for 30 min, followed by isolation of theDNA using miniprep column (Qiagen). Samples incubated under the samecondition, but with addition of H₂O instead of CAA were used as control.The isolated DNA was digested using ApaI (Fermentas Fastdigest) and thenwas the enzyme inactivated in 65° C. for 5 min.

Primer Extension:

Primer pMP1764F (5′-CTCTGGAGTAGCTGGGATTACAG-3′) and pMP1333R(5′-CCAACATGGTGAAACCCAGTATCTAC-3′) were 5′-labeled using [γ-³²P]ATP andT4 polynucleotide kinase (Fermentas) according to the manufacturersprotocol and then purified using QIAquick Nucleotide Removal Kit(QIAGEN). A primer extension mix (2 mM MgCl₂, 1 U taq polymerase(Fermentas), 5 nM primer, 2 mM of each dNTP), was added to approximately100 ng template (cleaved by CAA or BQQ-OP) and then exposed for thefollowing condition; 10 min at 94° C., 30 cycles of 1 min in 94° C., 2min in 54° C. or 49° C. (primer 1333R and 1764F, respectively) and 3 minin 72° C., and then finally 10 min in 72° C. As controls for the primerreaction were plasmids incubated under similar condition but withoutaddition of cleavage reagents (CAA, or BQQ-OP) used. PstI and Sadcleaved plasmid (100 ng) was used as templates for sequencing reactionusing dideoxynucleotides. The samples was then analyzed using denaturingpolyacrylamide gel electrophoresis (6%, 7 M urea, 0.5 mm) in buffer(1×TBE) at room temperature and 1200 V, 32 mA, 2.5 h. Fuji FLA3000phosphorimager was used for scanning, analysis and quantification.

Results Formation of PNA-directed Triplex at GAA Repeats

To investigate the ability of BQQ-derivatives to intercalate into aPNA-dsDNA triplex structure, BQQ-OP mediated triplex-specific cleavageof double strand DNA was employed. Plasmid pMP179 containing 115 GAArepeats flanked by intronic sequences of the frataxin gene waslinearized. The plasmid was then incubated with (CTT)_(n) PNA (3482),(GAA)_(n) PNA (3320) or (CTT)_(n) TFO (TFO standing for triplex formingoligonucleotide) in buffer (10 mM sodium cacodylate, pH 7.5, 100 mM NaCland 0 or 2 mM MgCl₂). PNA or TFO binding was analyzed using the BQQ-OPcleavage assay, which was analyzed using agarose gel electrophoresis.FIG. 3 (lanes 2 and 5) shows that an intermolecular triplex complex isformed in the presence of (CTT)_(n) PNA as indicated by the presence oftwo DNA fragments of the expected sizes (approximately 3814 and 3178 bp,assuming an average triplex cleavage in the middle of the GAA repeats).However, comparison of the amount of triplex-specific cleavage in thepresence of the (CTT)_(n) TFO (FIG. 3, lane 3 and 6) reveal that thecorresponding TFO-directed triplex, at the same target DNA, is morestable. More interestingly, formation of an intermolecular triplexstructure in the presence of the GAA-PNA was not detected (FIG. 3, lane1 and 4).

Sequence-Specific Interactions of PNA and H-DNA at GAA Repeats

It has recently been shown that BQQ-OP probe for the presence of anH-DNA structure formed at the 115 GAA repeats in supercoiled pMP179 (5).It was also demonstrated that the amount of inter- and intramoleculartriplex as detected by BQQ-OP mediated DNA double strand cleavage,significantly increases in the presence of CTT-TFO. On the other hand,addition of (GAA)_(n) TFO did not alter the amount of detected triplex.These results demonstrated that a pyrimidine motif H-DNA is the morestable motif formed at GAA repeats in plasmids. To examine the effectsof sequence-specific PNA binding of an H-DNA forming GAA repeats insupercoiled plasmids, PNA binding was performed followed by analysisusing the BQQ-OP cleavage assay. As reference the plasmid was incubatedwith the same concentration of (CTT)_(n)- and (GAA)_(n)-TFO or in theabsence of oligonucleotides. The result shows that addition of (CTT)_(n)PNA results in a clear increase in the amount of triplex formed (FIG. 4,lane 1 and 3). However, the effect is not fully as strong as in thepresence of (CTT)_(n)TFO (FIG. 4, lane 4). Interestingly, addition of(GAA)_(n) PNA prevented formation of an H-DNA structure, as detected byBQQ-OP cleavage (FIG. 4, lane 2). Taken together, the results indicatethat (CTT)_(n) PNA and (GAA)_(n) PNA binding to the H-DNA forming GAArepeats is highly sequence-specific and yields two different PNA-DNAcomplexes. Furthermore, (GAA)_(n) PNA binding interferes with H-DNAformation at FA associated repeats.

Affinity Cleavage of GAA Repeat Containing Plasmids:

To understand the structure formed at GAA repeats and the effect of PNA,(CTT)_(n) and (GAA)_(n), on H-DNA at this site, structure analysis wasconducted using chemical modifications by chloroacetaldeheyde (CAA) andtriplex-directed DNA double-strand cleavage using BQQ-OP. DNAmodification and cleavage were analyzed using primer extension reactionsand on denatured polyacrylamide gel electrophoresis. In agreement withprevious results, CAA cleavage of a plasmid containing short GAA repeats(9 repeats) (FIG. 5A) showed formation of a 3′3′5′ pyrimidine H-DNAstructure, leaving the 5′ end of the purine strand single stranded (FIG.5B). The BQQ-OP cleavage (FIG. 6A) confirms the CAA cleavage, showingthat the triplex is formed in the 3′end of the purine strand, it alsoshows that the part of the flanking region is involved in the triplexstructure.

When (CTT)_(n) PNA was added to this plasmid cleavage by BQQ-OP wasobserved in the 5′-end and 3′-end of the GAA and CTT strand,respectively (FIG. 6A). The CAA cleavage (FIG. 5) shows that thepyrimidine strand is single stranded, which indicate that a triplexinvasion complex is formed (FIG. 5). Similar cleavage pattern were alsoobserved after addition of a (GAA)_(n) PNA, although CAA cleavage weredetected on the purine strand (FIG. 5). The results indicate formationof an invasion complex by (GAA)_(n) PNA, which in this case correspondsto a duplex invasion (FIGS. 5 and 6).

Similar chemical modification and cleavage reactions were also performedat longer GAA repeats (75 repeats) (FIG. 7). In the absence of PNA, boththe purine and the pyrimidine strands were cleaved by BQQ-OP (FIG. 7),the cleavage is stronger in the 3′end of the purine strand indicatingthat the 3′3′5′isomer is predominant. Neither strand however showssingle strand modification by CAA (FIG. 7), which indicate the presenceof a more complex structure. After addition of a (CTT)_(n) PNA theBQQ-OP cleavage show a similar pattern as in the absence of PNA (FIG.7), although cleavage is much stronger. The cleavage is mainly found inthe 3′end of the purine strand and is clearly weaker in the 5′end. TheCAA cleavage clearly differs between the absence and presence of the(CTT)_(n) PNA, in the presence of (CTT)_(n) PNA the pyrimidine strandclearly is cleaved (FIG. 7) even though the same strand also is cleavedby BQQ-OP. The only explanation is that there exist several structuralconformations. At the shorter repeat, (CTT)_(n) PNA forms a triplexinvasion complex, seen as single strand cleavage of the pyrimidinestrand, similar to the cleavage seen at the longer repeats. However,BQQ-OP analysis (FIG. 3, lanes 2, 5) also shows that (CTT)_(n) PNA canform an intermolecular triplex structure that can be cleaved by BQQ-OP,thereby causing cleavage on both the purine and the pyrimidine strand.In conclusion, addition of (CTT)_(n) PNA to the plasmid containinglonger repeat (75 repeats) results in the formation of triplex invasionsand intermolecular DNA-PNA complexes.

In the presence of (GAA)_(n) PNA, no BQQ-OP directed cleavage could bedetected either when analyzed on agarose gel (FIG. 3) (FIG. 4) or afterprimer extension (FIG. 7). On the other hand, chemical modificationusing CAA revealed that a single strand purine strand is predominant(FIG. 7). This result demonstrates that the (GAA), PNA forms a duplexinvasion complex, and that formation of this DNA-PNA complex preventsintramolecular (H-DNA) formation at FA GAA repeat expansions.

Application of (CTT)_(n) and (GAA)_(n) PNA in FA Diagnostics

The sequence-specific binding of (CTT)_(n) PNA and (GAA)_(n) PNA whichleads to the formation of distinguishable DNA-PNA complexes and preventsintramolecular DNA triplex formation within higher order DNA structuresat FA pathological GAA repeat expansions has two main applications:First, (GAA)_(n) PNA dissociate triplex structures and can be used inPCR mediated amplification, primer extension (PE), Southern blotanalysis or DNA sequencing of frataxin GAA repeats including short,medium and large repeats. Second, (CTT)_(n) PNA forms intermoleculartriplex and triplex invasion DNA complexes and can be used in PCRmediated amplification, primer extension, Southern blot analysis or DNAsequencing of frataxin GAA repeats including short, medium and largerepeats. Third, labeled (CTT)_(n) and (GAA)_(n) PNA can be used eitherin direct detection of PCR or PE fragments or as sequence-specificprobes to be used in Southern blot. (CTT)_(n) and (GAA)_(n) PNA act thenthrough a sequence-specific targeting of DNA structures through bindingto double strand and/or single strand DNA.

Example 2 Analysis of a Blood Sample for the Diagnosis of Friedreich'sAtaxia

-   -   1. A blood sample will be collected from a Friedreich's ataxia        potential carrier or patient.    -   2. Isolation of total DNA from the sample will be carried out        according to a standard protocol using a commercially available        kit for DNA isolation, such as the “DNA isolation kit for cells        and tissues”, Roche Applied Sciences”.    -   3. A (GAA)_(n) PNA (21 residues) having a 6 nt flanking sequence        that will be composed of *substantially non-complementary        deoxyoligonucleotides will be dissolved in water, and the        solution will be heated at 95° C. for 3 min and kept directly on        ice. * Substantially non-complementary means that the sequence        has a hybridization T_(m)≦37° C. with the GAA or CTT repeat.    -   4. The oligonucleotide (from step 3) will be added to the        isolated DNA (from step 2) in a ratio of DNA:oligo, 1:500 in a        buffer (10 mM Tris-Hcl. 140 mM KCl, pH 7.5). The mixture will be        incubated at 37° C. for 60 min.    -   5. The GAA repeat and flanking regions (322 bp) will be        amplified using a pair of primers (23 residues) complementary to        the 5′ and 3′flanking region of the GAA repeats in intron 1 of        the frataxin gene. Amplification will be carried out using a        thermal cycler and a polymerase chain reaction according to        standard protocol.    -   6. A Cy5-labeled (CTT)_(n) PNA (15 residues, dissolved in water)        will be heated at 45° C. for 3 min and kept directly on ice.    -   7. The PCR reaction mixture in buffer (10 mM Tris-HCl, pH 8.3,        50 mM KCl, 1.5 mM MgCl₂, 0.001 Gelatin) will be hybridized to        the Cy5-labeled (CTT)_(n) PNA (from step 6) in a ratio of        DNA:oligo, 1:10000. The mixture will be incubated at 37° C. for        60 min.    -   8. The mixture (from step 7) will be loaded on a 0.7% agarose        gel in 0.5×TBE. The gel will be run in 0.5×TBE, 50V, 1.5 h. A        DNA molecular weight ladder (containing ethidium bromide) will        be loaded on the same gel.    -   9. The labeled PCR fragments (Cy5-(CTT)_(n)-PNA labeled) will be        visualized using a phosphoimager (fluorescence reading        λ_(ex)=550 nm and λ_(em)=570 nm) and the size of the obtained        labeled PCR fragments will be compared to the DNA ladder.    -   10. The gel will be stained with ethidium bromide (0.1 μg/ml in        0.5×TBE).    -   11. The DNA separated on the gel (from step 10) will be        visualized using a GelDoc.    -   12. The size of the amplified GAA repeat containing fragments on        the gel (step11) will also be compared to the DNA ladder.    -   13. The number of the GAA repeats will be determined by the        following equation: size of fragment=322+3n.    -   14. Two PCR fragments of different sizes are expected to be        obtained. In the case where both fragments are in the range of        pathological expansions (90-1700 repeats) the results are        interpreted as belonging to a potential Friedreich's ataxia        patient. In other cases where the size of one or both fragments        is in the range of pre-mutated or normal GAA repeats, the        results are interpreted as belonging to a carrier or a healthy        individual, respectively.

REFERENCES

-   1. Campuzano, V., Montermini, L., Molto, M. D., Pianese, L., Cossee,    M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F.,    Monticelli, A. et al. (1996) Friedreich's ataxia: autosomal    recessive disease caused by an intronic GAA triplet repeat    expansion. Science (New York, N.Y., 271, 1423-1427.-   2. Campuzano, V., Montermini, L., Lutz, Y., Cova, L., Hindelang, C.,    Jiralerspong, S., Trottier, Y., Kish, S. J., Faucheux, B.,    Trouillas, P. et al. (1997) Frataxin is reduced in Friedreich ataxia    patients and is associated with mitochondrial membranes. Human    molecular genetics, 6, 1771-1780.-   3. Pandolfo, M. (2002) Frataxin deficiency and mitochondrial    dysfunction. Mitochondrion, 2, 87-93.-   4. Sakamoto, N., Chastain, P. D., Parniewski, P., Ohshima, K.,    Pandolfo, M., Griffith, J. D. and Wells, R. D. (1999) Sticky DNA:    self-association properties of long GAA.TTC repeats in R.R.Y triplex    structures from Friedreich's ataxia. Molecular cell, 3, 465-475.-   5. Bergquist, H., Nikravesh, A., Fernandez, R. D., Larsson, V.,    Nguyen, C. H., Good, L., and Zain, R. (2009). Structure-specific    recognition of Friedreich's ataxia (GAA)n repeats by    benzoquinoquinoxaline derivatives. ChemBioChem 10, 2629-2637.-   6. Napierala, M., Dere, R., Vetcher, A. and Wells, R. D. (2004)    Structure-dependent recombination hot spot activity of GAA.TTC    sequences from intron 1 of the Friedreich's ataxia gene. The Journal    of biological chemistry, 279, 6444-6454.-   7. Krasilnikova, M. M. and Mirkin, S. M. (2004) Replication stalling    at Friedreich's ataxia (GAA)n repeats in vivo. Mol. Cell. Biol., 24,    2286-2295.-   8. Krasilnikova, M. M., Kireeva, M. L., Petrovic, V., Knijnikova,    N., Kashlev, M. and Mirkin, S. M. (2007) Effects of Friedreich's    ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability. Nucleic    acids research, 35, 1075-1084.-   9. Nielsen, P. E., Egholm, M., Berg, R. H. and Buchardt, O. (1991)    Sequence-selective recognition of DNA by strand displacement with a    thymine-substituted polyamide. Science (New York, N.Y., 254,    1497-1500.-   10. Bentin, T., Hansen, G. I. and Nielsen, P. E. (2006) Structural    diversity of target-specific homopyrimidine peptide nucleic    acid-dsDNA complexes. Nucleic acids research, 34, 5790-5799.-   11. WO 2008/018795 A1, Prosenza B. V. et al. (2008).-   12. Grabczyk E. And Usdin K., (2000) Alleviating transcript    insufficiency caused by Friedreich's ataxia repeats. Nucleic acids    research, 28, 4930-4937.

1-19. (canceled)
 20. A method for diagnosing Friedreich's ataxia in asubject comprising the steps of: a) isolating DNA, which is genomic DNA,from a biological sample, b) optionally cleaving the DNA isolated instep a) with one or more DNA restriction enzyme(s), c) adding anoligonucleotide, said oligonucleotide consisting of a sequence selectedfrom the group consisting of (GAA)_(n), (CTT)_(n), (JTT)_(n), or a mixed(JTT/CTT)_(n) sequence, wherein n is about 2-10, and whereinoligonucleotide is based on a peptide nucleic acid oligonucleotide or anequivalent oligonucleotide analogue, such as morpholino oligonucleotideor a locked nucleic acid oligonucleotide, to the isolated DNA of stepa), or the cleaved DNA of step b), d) dissociating and/or abolishing theformation of higher order DNA structures, such as H-DNA and triplexformation, at GAA repeats, and e) determining the length, sequenceand/or number of GAA repeats in the frataxin gene.
 21. A methodaccording to claim 20, wherein the oligonucleotide consists of asequence selected from the group consisting of (GAA)_(n), (CTT)_(n) or(JTT)_(n) sequence.
 22. A method according to claim 20, wherein theoligonucleotide consists of a (GAA)_(n) sequence, wherein theoligonucleotide is based on a peptide nucleic acid oligonucleotide or anequivalent oligonucleotide analogue, such as morpholino or a lockednucleic acid.
 23. A method according to claim 20, wherein saidoligonucleotide is (GAA)_(n) based on peptide nucleic acid.
 24. A methodaccording to claim 20, wherein the oligonucleotide consists of a(CTT)_(n) sequence, wherein the oligonucleotide is based on a peptidenucleic acid oligonucleotide or an equivalent oligonucleotide analogue,such as morpholino or a locked nucleic acid.
 25. A method according toclaim 20, wherein the oligonucleotide consists of a (JTT)_(n) sequence,wherein the oligonucleotide is based on a peptide nucleic acidoligonucleotide or an equivalent oligonucleotide analogue, such asmorpholino or a locked nucleic acid.
 26. A method according to claim 20,wherein the oligonucleotide is Ac-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,H-LysLys-GAAGAAGAAGAA-Lys-NH₂, Ac-TTCTTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂,Acr-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,Ac-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,Ac-TTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂, H-LysLys-GAAGAAGAAGAAGAA-Lys-NH₂,H-LysLys-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂, Acr-eg1-GAAGAAGAAGAA-Lys-NH₂,Acr-eg1-GAAGAAGAAGAAGAA-Lys-NH₂, Acr-eg1-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂.27. The method according to claim 20, wherein the oligonucleotide ispeptide nucleic acid, morpholino or locked nucleic acid.
 28. The methodaccording to claim 20, wherein the number of residues in theoligonucleotide is 6-30, or 12-21.
 29. The method according to claim 20,wherein the oligonucleotide has N- and C-terminal chemical groups,wherein the chemical groups are eg1=ethylene glycol linker, Lys=Lysine,Ac=acetyl, Acr=acridine, diMeLys=dimethyl lysine.
 30. A method accordingto claim 20, wherein said sequence further comprises a terminal flankingsequence in one or both ends of said oligonucleotide.
 31. A methodaccording to claim 20, wherein step e) comprises performing a polymerasechain reaction, primer extension reaction, DNA sequencing or Southernblotting.
 32. Use of oligonucleotide as defined in claim 20, fordissociating and/or abolishing the formation of higher order structuresin genomic DNA, such as triplex formation, at GAA repeats, enabling acorrect functioning and/or expression of the frataxin gene.
 33. The useaccording to claim 32, wherein the oligonucleotide consists of asequence selected from the group consisting of (GAA)_(n), (CTT)_(n) or(JTT)_(n) sequence.
 34. The use according to claim 32, wherein theoligonucleotide consists of a (GAA)_(n) sequence, wherein theoligonucleotide is based on a peptide nucleic acid oligonucleotide or anequivalent oligonucleotide analogue, such as morpholino or a lockednucleic acid.
 35. The use according to claim 32, wherein saidoligonucleotide is (GAA)_(n) based on peptide nucleic acid.
 36. The useaccording to claim 32, wherein the oligonucleotide consists of a(CTT)_(n) sequence, wherein the oligonucleotide is based on a peptidenucleic acid oligonucleotide or an equivalent oligonucleotide analogue,such as morpholino or a locked nucleic acid.
 37. The use according toclaim 32, wherein the oligonucleotide consists of a (JTT)_(n) sequence,wherein the oligonucleotide is based on a peptide nucleic acidoligonucleotide or an equivalent oligonucleotide analogue, such asmorpholino or a locked nucleic acid.
 38. The use according to claim 32,wherein the oligonucleotide is Ac-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,H-LysLys-GAAGAAGAAGAA-Lys-NH₂, Ac-TTCTTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂,Acr-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,Ac-(diMeLys)₂-TTCTTCTTCTTCTTC-eg1-Lys-NH₂,Ac-TTCTTCTTCTTCTTCTTC-eg1-Lys-NH₂, H-LysLys-GAAGAAGAAGAAGAA-Lys-NH₂,H-LysLys-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂, Acr-eg1-GAAGAAGAAGAA-Lys-NH₂,Acr-eg1-GAAGAAGAAGAAGAA-Lys-NH₂, Acr-eg1-GAAGAAGAAGAAGAAGAAGAA-Lys-NH₂.39. The use according to claim 32, wherein said sequence furthercomprises a terminal flanking sequence in one or both ends of saidoligonucleotide.
 40. The use according to claim 32, wherein the numberof residues in the oligonucleotide is 6-30, or 12-21.
 41. The useaccording to claim 32, for prevention and/or treatment of Friedreich'sataxia.
 42. The use according to claim 32, for determining the lengthand/or number of GAA repeats in a repeated GAA sequence, such as in thefrataxin gene.
 43. The use according to claim 32, wherein theoligonucleotide is comprised in a pharmaceutical composition, optionallyin combination with a pharmaceutically acceptable carrier, adjuvantand/or excipient.